Compositions and Methods for Treatment of Cardiovascular Disorders

ABSTRACT

ECM based compositions including amniotic membrane and methods for employing same to treat cardiovascular disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/782,115, filed on Mar. 1, 2013, which is a continuation-in-part ofU.S. application Ser. No. 13/573,569, filed on Sep. 24, 2012, which is acontinuation-in-part of U.S. application Ser. No. 11/334,631, filed onJan. 18, 2006, which is a continuation of application Ser. No.12/371,158, filed on Feb. 13, 2009, now abandoned, which is acontinuation of application Ser. No. 11/747,018, filed on May 10, 2007,now abandoned.

FIELD OF THE INVENTION

The present invention relates to methods for treating cardiovasculardisorders. More particularly, the present invention relates toextracellular matrix (ECM) compositions and methods for treatingcardiovascular disorders.

BACKGROUND OF THE INVENTION

As is well known in the art, heart failure can be caused by a diversearray of cardiovascular disorders that reduce the efficiency of themyocardium, including ischemic heart disease, coronary artery disease,and a defective or diseased heart valve. Among the noted disorders,ischemic heart disease, which commonly presents as a myocardialinfarction, is the leading cause of heart failure.

Indeed, in 2004 alone, the World Health Organization estimated that12.2% of worldwide deaths occurred as a result of ischemic heartdisease. Ischemic heart disease was also deemed the leading cause ofdeath in middle to high income countries and second only to respiratoryinfections in lower income countries. The Global Burden of Disease:World Health Organization 2004 Update, Geneva (2008). Worldwide morethan 3 million people present with a ST elevation myocardial infarction(STEMI) and 4 million people present with a non-ST elevation myocardialinfarction (NSTEMI) a year. White, et al., Acute Myocardial Infarction,Lancet 372 (9638), pp. 570-84 (August 2008).

Rates of death from ischemic heart disease have slowed or declined inmost high income countries, although cardiovascular disease stillaccounted for 1 in 3 of all deaths in the USA in 2008. Roger, et al.,Executive summary: Heart Disease and Stroke Statistics-2012 update: Areport from the American Heart Association, Circulation 125 (1), pp.188-97 (January 2012).

In contrast, ischemic heart disease is becoming a more common cause ofdeath in the developing world. For example in India, ischemic heartdisease had become the leading cause of death by 2004; accounting for1.46 million deaths (14% of total deaths). Deaths in India due toischemic heart disease were also expected to double during 1985-2015.Gupta, et al., Epidemiology and Causation of Coronary Heart Disease andStroke in India, Heart 94 (1), pp. 16-26 (January 2008).

Globally, it is predicted that disability adjusted life years (DALYs)lost to ischemic heart disease will account for 5.5% of total DALYs in2030, making it the second most important cause of disability (afterunipolar depressive disorder), as well as the leading cause of death bythis date.

Ischemic heart disease often occurs when myocardial tissue is no longerreceiving adequate blood flow. Various methods for treating ischemicheart disease have thus been developed. Such methods include systemicdelivery of various pharmacological agents.

Several additional methods for treating ischemic heart disease aredirected to re-establishing blood flow to the ischemic area. Suchmethods include stimulation of angiogenesis and surgical intervention,e.g. bypass surgery or angioplasty. Other methods include the use oflasers to bore holes through the ischemic area(s) to promote blood flow.As one can readily appreciate, there are numerous incumbent risksassociated with the noted methods.

A further method for treating ischemic heart disease is the directdelivery of bioactive or pharmacological agents to the ischemic area.Illustrative is the delivery of extracellular matrix (ECM) basedcompositions directly to cardiovascular tissue disclosed in Co-pendingapplication Ser. No. 13/573,569.

More recently, ventricular assist devices (VADs) have been employed astreatment platforms for various pharmacological therapies, e.g. stemcell administration, which have been developed to treat cardiovasculardisorders, including ischemic heart disease. VADs are designed tosupport (or augment) the function of either the right (RVAD) or left(LVAD) ventricle, or both at once (BiVAD). The type of VAD employeddepends primarily on the underlying cardiovascular disorder, and thepulmonary arterial resistance that determines the load on the rightventricle.

Although the direct delivery of bioactive or pharmacological agents;particularly, the ECM based compositions disclosed in Co-pendingapplication Ser. No. 13/573,569, and other treatment therapies employingventricular assistance have been found effective to treat cardiovasculardisorders and, thereby, heart failure, there remains a need to provideeven more effective means for treating cardiovascular disorders.

It is therefore an object of the present invention to provide improvedcompositions and methods for treating damaged or diseased biologicaltissue; particularly, cardiovascular tissue and, hence, cardiovasculardisorders associated therewith.

It is another object of the present invention to provide ECM basedcompositions comprising amniotic membrane for treating damaged ordiseased biological tissue.

It is another an object of the present invention to provide ECM basedcompositions that promote tissue survival, and induce neovascularizationand regeneration of damaged cardiovascular tissue.

It is yet another object of the present invention to provide methods fortreating cardiovascular disorders that includes administration of an ECMbased composition, which, when delivered to damaged biological tissue;particularly, cardiovascular tissue, induces neovascularization, hosttissue proliferation, bioremodeling, and regeneration of cardiovasculartissue and associated structures with site-specific structural andfunctional properties.

SUMMARY OF THE INVENTION

The present invention is directed to ECM based compositions and methodsfor treating damaged or diseased biological tissue.

In a preferred embodiment of the invention, the ECM based compositionscomprise amniotic membrane.

In some embodiments, the ECM based compositions further include at leastone additional biologically active agent that supports the treatment ofdamaged cardiovascular tissue and/or bioremodeling and/or regenerationof tissue.

In some embodiments of the invention, the biologically active agentcomprises a growth factor selected from the group comprisingtransforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), fibroblast growth factor-2 (FGF-2), basicfibroblast growth factor (bFGF) and vascular epithelial growth factor(VEGF).

In some embodiments of the invention, the biologically active agentcomprises a cell selected from the group comprising human embryonic stemcells, fetal cardiomyocytes, myofibroblasts, and mesenchymal stem cells.

In some embodiments of the invention, the biologically active agentcomprises a protein selected from the group comprising proteoglycans,glycosaminoglycans (GAGs), glycoproteins and cytokines.

In some embodiments, the ECM based compositions further include at leastone pharmacological agent.

In some embodiments of the invention, the pharmacological agentcomprises an antiarrhythmic agent selected from the group comprisingquinidine, procainamide, disopyramide, lidocaine, phenytoin, mexiletine,flecainide, propafenone, moricizine, propranolol, esmolol, timolol,metoprolol, atenolol, amiodarone, sotalol, ibutilide, dofetilide,verapamil, diltiazem, adenosine and digoxin.

In some embodiments of the invention, the pharmacological agentcomprises an anti-inflammatory agent or composition.

In some embodiments of the invention, the biologically active componentcomprises a statin selected from the group comprising atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, and simvastatin.

In some embodiments of the invention, the ECM based compositions areformulated to facilitate injection of the ECM based compositions todamaged or diseased tissue (i.e. injectable compositions).

In some embodiments of the invention, ECM based compositions are in theform of a graft.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages will become apparent from the followingand more particular description of the preferred embodiments of theinvention, as illustrated in the accompanying drawings, and in whichlike referenced characters generally refer to the same parts or elementsthroughout the views, and in which:

FIG. 1 is a depiction of a normal heart;

FIG. 2 is a of a heart having an ischemic infracted region;

FIG. 3A is an exploded perspective view of one embodiment of amulti-needle injection apparatus that is suitable for directadministration of ECM compositions to biological tissue, e.g.cardiovascular tissue, in accordance with the invention; and

FIG. 3B is an assembled perspective view of the multi-needle injectionapparatus shown in FIG. 3A, in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified apparatus, systems, compositions or methods as such may, ofcourse, vary. Thus, although a number of systems, compositions andmethods similar or equivalent to those described herein can be used inthe practice of the present invention, the preferred systems,compositions and methods are described herein.

It is also to be understood that, although a preferred method ofdelivering an ECM based composition of the invention to biologicaltissue comprises direct injection into the tissue. The delivery of anECM based composition is not limited to direct injection. According tothe invention, an ECM based composition of the invention can bedelivered to biological tissue by other conventional means, includingtopical administration.

It is further to be understood that the terminology used herein is forthe purpose of describing particular embodiments of the invention onlyand is not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which the invention pertains.

Further, all publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

Finally, as used in this specification and the appended claims, thesingular forms “a, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “ananti-inflammatory” includes two or more such agents and the like.

DEFINITIONS

The terms “cardiovascular disorder” and “heart failure” are usedinterchangeably herein, and mean and include any abnormal function ofthe heart; particularly, abnormal functions or deficiency of themyocardium. The terms “cardiovascular disorder” and “heart failure” thusinclude, without limitation, ischemic heart disease, coronary arterydisease, a defective or diseased heart valve, myocarditis, aninflammatory disease, cardiomyopathy and amyloidosis.

The terms “cardiovascular tissue damage,” “cardiac tissue damage,” and“cardiac tissue injury” and are used interchangeably herein, and meanand include any area of abnormal tissue in the cardiovascular system orheart caused by a disease, disorder, injury or damage, including damageto the epicardium, endocardium and/or myocardium.

As is well known in the art, cardiovascular tissue damage most ofteninvolves damage or injury to the myocardium and, therefore, for thepurposes of this disclosure, myocardial damage or injury is equivalentto cardiovascular tissue damage.

The terms “extracellular matrix”, “ECM” and “ECM material” are usedinterchangeably herein, and mean and include a collagen-rich substancethat is found in between cells in mammalian tissue, and any materialprocessed therefrom, e.g. decellularized ECM. According to theinvention, ECM can be derived from a variety of mammalian tissuesources, including, without limitation, small intestine submucosa (SIS),urinary bladder submucosa (UBS), stomach submucosa (SS), central nervoussystem tissue, epithelium of mesodermal origin, i.e. mesothelial tissue,dermal extracellular matrix, subcutaneous extracellular matrix,gastrointestinal extracellular matrix, i.e. large and small intestines,tissue surrounding growing bone, placental extracellular matrix, omentumextracellular matrix, cardiac extracellular matrix, e.g., pericardiumand/or myocardium, kidney extracellular matrix, pancreas extracellularmatrix, lung extracellular matrix, and combinations thereof. The ECMmaterial can also comprise collagen from mammalian sources.

ECM can also be derived from basement membrane of mammaliantissue/organs, including, without limitation, urinary basement membrane(UBM), liver basement membrane (LBM), and amnion, chorion, allograftpericardium, allograft acellular dermis, amniotic membrane, Wharton'sjelly, and combinations thereof.

The term “chamber remodeling”, as used herein, means and includes aseries of events (which may include changes in gene expression,molecular, cellular and interstitial changes) that result in changes insize, shape and function of biological tissue following stress orinjury. As is well known in the art, remodeling can occur after amyocardial infarction, pressure overload (e.g., aortic stenosis,hypertension), volume overload (e.g., valvular regurgitation),inflammatory heart disease (e.g., myocarditis), or in idiopathic cases(e.g., idiopathic dilated cardiomyopathy).

The term “angiogenesis”, as used herein, means a physiologic processinvolving the growth of new blood vessels from pre-existing bloodvessels.

The term “neovascularization”, as used herein, means and includes theformation of functional vascular networks that can be perfused by bloodor blood components. Neovascularization includes angiogenesis, buddingangiogenesis, intussuceptive angiogenesis, sprouting angiogenesis,therapeutic angiogenesis and vasculogenesis.

The terms “pharmacological agent”, “pharmacological composition” and“biologically active agent”, as used herein, mean and include an agent,drug, compound, composition of matter or mixture thereof, including itsformulation, which provides some therapeutic, often beneficial, effect.This includes any physiologically or pharmacologically active substancethat produces a localized or systemic effect or effects in animals,including warm blooded mammals, humans and primates; avians; domestichousehold or farm animals, such as cats, dogs, sheep, goats, cattle,horses and pigs; laboratory animals, such as mice, rats and guinea pigs;fish; reptiles; zoo and wild animals; and the like.

The terms “pharmacological agent” and “biologically active agent” thusmean and include, without limitation, antibiotics, anti-arrhythmicagents, anti-viral agents, analgesics, steroidal anti-inflammatories,non-steroidal anti-inflammatories, anti-neoplastics, anti-spasmodics,modulators of cell-extracellular matrix interactions, proteins,hormones, growth factors, matrix metalloproteinases (MMPS), enzymes andenzyme inhibitors, anticoagulants and/or antithrombic agents, DNA, RNA,modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or proteinsynthesis, polypeptides, oligonucleotides, polynucleotides,nucleoproteins, compounds modulating cell migration, compoundsmodulating proliferation and growth of tissue, and vasodilating agents.

The terms “pharmacological agent” and “biologically active agent”accordingly include, without limitation, atropine, tropicamide,dexamethasone, dexamethasone phosphate, betamethasone, betamethasonephosphate, prednisolone, triamcinolone, triamcinolone acetonide,fluocinolone acetonide, anecortave acetate, budesonide, cyclosporine,FK-506, rapamycin, ruboxistaurin, midostaurin, flurbiprofen, suprofen,ketoprofen, diclofenac, ketorolac, nepafenac, lidocaine, neomycin,polymyxin b, bacitracin, gramicidin, gentamicin, oyxtetracycline,ciprofloxacin, ofloxacin, tobramycin, amikacin, vancomycin, cefazolin,ticarcillin, chloramphenicol, miconazole, itraconazole, trifluridine,vidarabine, ganciclovir, acyclovir, cidofovir, ara-amp, foscarnet,idoxuridine, adefovir dipivoxil, methotrexate, carboplatin,phenylephrine, epinephrine, dipivefrin, timolol, 6-hydroxydopamine,betaxolol, pilocarpine, carbachol, physostigmine, demecarium,dorzolamide, brinzolamide, latanoprost, sodium hyaluronate, insulin,verteporfin, pegaptanib, ranibizumab, and other antibodies,antineoplastics, Anti VGEFs, ciliary neurotrophic factor, brain-derivedneurotrophic factor, bFGF, Caspase-1 inhibitors, Caspase-3 inhibitors,α-Adrenoceptors agonists, NMDA antagonists, Glial cell line-derivedneurotrophic factors (GDNF), pigment epithelium-derived factor (PEDF),and NT-3, NT-4, NGF, IGF-2.

According to the invention, the terms “pharmacological agent” and“biologically active agent” further include, without limitation, thefollowing growth factors: platelet derived growth factor (PDGF),epidermal growth factor (EGF), transforming growth factor alpha(TGF-alpha), transforming growth factor beta (TGF-beta), fibroblastgrowth factor-2 (FGF-2), basic fibroblast growth factor (bFGF), vascularepithelial growth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), nerve growth factor (NGF), plateletderived growth factor (PDGF), tumor necrosis factor alpha (TNA-alpha),and placental growth factor (PLGF).

The terms “pharmacological agent” and “biologically active agent”further include, without limitation, the following Class I-Class Vantiarrhythmic agents: (Class Ia) quinidine, procainamide anddisopyramide; (Class Ib) lidocaine, phenytoin and mexiletine; (Class Ic)flecainide, propafenone and moricizine; (Class II) propranolol, esmolol,timolol, metoprolol and atenolol; (Class III) amiodarone, sotalol,ibutilide and dofetilide; (Class IV) verapamil and diltiazem) and (ClassV) adenosine and digoxin.

The terms “pharmacological agent” and “biologically active agent”further include, without limitation, the following anti-inflammatories:alclofenac, alclometasone dipropionate, algestone acetonide, alphaamylase, amcinafal, amcinafide, amfenac sodium, amiprilosehydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazidedisodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains,broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen,clobetasol propionate, clobetasone butyrate, clopirac, cloticasonepropionate, cormethasone acetate, cortodoxone, decanoate, deflazacort,delatestryl, depo-testosterone, desonide, desoximetasone, dexamethasonedipropionate, diclofenac potassium, diclofenac sodium, diflorasonediacetate, diflumidone sodium, diflunisal, difluprednate, diftalone,dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium,epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen,fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone,fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin,flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, mesterolone,methandrostenolone, methenolone, methenolone acetate, methylprednisolonesuleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxensodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin,oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranylinehydrochloride, pentosan polysulfate sodium, phenbutazone sodiumglycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate,talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam,tesimide, testosterone, testosterone blends, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, and zomepirac sodium.

The term “biologically active agent” further includes, withoutlimitation, organisms that have the potential to induce modulatingproliferation, and/or growth and/or regeneration of tissue. The terms“biologically active agent” thus includes, without limitation, thefollowing cells: human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, autotransplated expandedcardiomyocytes, adipocytes, totipotent cells, pluripotent cells, bloodstem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymalcells, embryonic stem cells, parenchymal cells, epithelial cells,endothelial cells, mesothelial cells, fibroblasts, osteoblasts,chondrocytes, exogenous cells, endogenous cells, stem cells,hematopoietic stem cells, bone-marrow derived progenitor cells,myocardial cells, skeletal cells, fetal cells, undifferentiated cells,multi-potent progenitor cells, unipotent progenitor cells, monocytes,cardiac myoblasts, skeletal myoblasts, macrophages, capillaryendothelial cells, xenogenic cells, allogenic cells, and post-natal stemcells.

According to the invention, the terms “pharmacological agent” and“biologically active agent” can further include the following activeagents (referred to interchangeably herein as a “protein”, “peptide” and“polypeptide”): collagen (types I-V), proteoglycans, glycosaminoglycans(GAGs), glycoproteins, cytokines, cell-surface associated proteins, celladhesion molecules (CAM), angiogenic growth factors, endothelialligands, matrikines, cadherins, immuoglobins, fibril collagens,non-fibrallar collagens, basement membrane collagens, multiplexins,small-leucine rich proteoglycans, decorins, biglycans, fibromodulins,keratocans, lumicans, epiphycans, heparin sulfate proteoglycans,perlecans, agrins, testicans, syndecans, glypicans, serglycins,selectins, lecticans, aggrecans, versicans, neurocans, brevicans,cytoplasmic domain-44 (CD-44), macrophage stimulating factors, amyloidprecursor proteins, heparins, chondroitin sulfate B (dermatan sulfate),chondroitin sulfate A, heparin sulfates, hyaluronic acids, fibronectins,tenascins, elastins, fibrillins, laminins, nidogen/enactins, fibulin I,finulin II, integrins, transmembrane molecules, thrombospondins,ostepontins, and angiotensin converting enzymes (ACE).

The terms “ECM composition” and “ECM based composition” are usedinterchangeably herein and mean and include a composition comprising atleast one ECM material, and, in some embodiments; preferably amnioticmembrane, and/or a “biologically active agent” and/or “pharmacologicalagent” and/or any additional agent or component identified herein.

The term “therapeutically effective”, as used herein, means that theamount of the “ECM composition” and/or “ECM based composition” and/or“biologically active agent” and/or “pharmacological agent” administeredis of sufficient quantity to ameliorate one or more causes, symptoms, orsequelae of a disease or disorder. Such amelioration only requires areduction or alteration, not necessarily elimination, of the cause,symptom, or sequelae of a disease or disorder.

The terms “prevent” and “preventing” are used interchangeably herein,and mean and include reducing the frequency or severity of a disease,condition or disorder. The term does not require an absolute preclusionof the disease, condition or disorder. Rather, this term includesdecreasing the chance for disease occurrence.

The terms “treat” and “treatment” are used interchangeably herein, andmean and include medical management of a patient with the intent tocure, ameliorate, stabilize, or prevent a disease, pathologicalcondition or disorder. The terms include “active treatment”, i.e.treatment directed specifically toward the improvement of a disease,pathological condition or disorder, and “causal treatment”, i.e.treatment directed toward removal of the cause of the associateddisease, pathological condition or disorder.

The terms “treat” and “treatment” further include “palliativetreatment”, i.e. treatment designed for the relief of symptoms ratherthan the curing of the disease, pathological condition or disorder,“preventative treatment”, i.e. treatment directed to minimizing orpartially or completely inhibiting the development of the associateddisease, pathological condition or disorder, and “supportive treatment”,i.e. treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathological conditionor disorder.

The terms “delivery” and “administration” are used interchangeablyherein, and mean and include providing an “ECM composition” or “ECMbased composition” to a treatment site, e.g., damaged tissue, throughany method appropriate to deliver the functional agent or formulation orcomposition to the treatment site. Non-limiting examples of deliverymethods include direct injection, percutaneous delivery and topicalapplication at the treatment site.

The term “percutaneous”, as used herein, means and includes anypenetration through the skin of a patient or subject, whether in theform of a small cut, incision, hole, cannula, tubular access sleeve orport or the like.

The terms “patient” and “subject” are used interchangeably herein, andmean and include warm blooded mammals, humans and primates; avians;domestic household or farm animals, such as cats, dogs, sheep, goats,cattle, horses and pigs; laboratory animals, such as mice, rats andguinea pigs; fish; reptiles; zoo and wild animals; and the like.

The term “comprise” and variations of the term, such as “comprising” and“comprises,” means “including, but not limited to” and is not intendedto exclude, for example, other additives, components, integers or steps.

The following disclosure is provided to further explain in an enablingfashion the best modes of performing one or more embodiments of thepresent invention. The disclosure is further offered to enhance anunderstanding and appreciation for the inventive principles andadvantages thereof, rather than to limit in any manner the invention.The invention is defined solely by the appended claims including anyamendments made during the pendency of this application and allequivalents of those claims as issued.

As indicated above, the present invention is directed to ECM basedcompositions and methods for treating damaged or diseased biologicaltissue.

In a preferred embodiment of the invention, the ECM based compositionsinclude at least one extracellular matrix (hereinafter “ECM material”).

According to the invention, the ECM material can be derived from variousmammalian tissue sources and methods for preparing same, such asdisclosed in U.S. Pat. Nos. 7,550,004, 7,244,444, 6,379,710, 6,358,284,6,206,931, 5,733,337 and 4,902,508 and U.S. application Ser. No.12/707,427; which are incorporated by reference herein in theirentirety. The mammalian tissue sources include, without limitation,small intestine submucosa (SIS), urinary bladder submucosa (UBS),stomach submucosa (SS), central nervous system tissue, epithelium ofmesodermal origin, i.e. mesothelial tissue, dermal extracellular matrix,subcutaneous extracellular matrix, gastrointestinal extracellularmatrix, i.e. large and small intestines, tissue surrounding growingbone, placental extracellular matrix, ornamentum extracellular matrix,cardiac extracellular matrix, e.g., pericardium and/or myocardium,kidney extracellular matrix, pancreas extracellular matrix, lungextracellular matrix, and combinations thereof. The ECM material canalso comprise collagen from mammalian sources.

The ECM material can also be derived from basement membrane of mammaliantissue/organs, including, without limitation, urinary basement membrane(UBM), liver basement membrane (LBM), and amnion, chorion, allograftpericardium, allograft acellular dermis, amniotic membrane, Wharton'sjelly, and combinations thereof.

In a preferred embodiment of the invention, the ECM based compositionscomprise amniotic membrane.

According to the invention, the ECM based compositions can comprisemixed liquids, mixed emulsions, mixed gels, mixed pastes, or mixed solidparticulates. The ECM based compositions can also comprise solidmembers, such as grafts.

According to the invention, the ECM based compositions of the inventioncan further include one or more biologically active or pharmacologicalagents or compositions, which aid in the treatment of damaged tissueand/or facilitate the bioremodeling and/or tissue regeneration process.

In some embodiments of the invention, the biologically active agentcomprises a growth factor. According to the invention, suitable growthfactors include, without limitation, a platelet derived growth factor(PDGF), epidermal growth factor (EGF), transforming growth factor alpha(TGF-alpha), transforming growth factor beta (TGF-beta), fibroblastgrowth factor-2 (FGF-2), basic fibroblast growth factor (bFGF), vascularepithelial growth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), nerve growth factor (NGF), plateletderived growth factor (PDGF), tumor necrosis factor alpha (TNA-alpha),and placental growth factor (PLGF).

In some embodiments of the invention, the biologically active agentcomprises a cell. According to the invention, suitable cells include,without limitation, a human embryonic stem cell, fetal cardiomyocyte,myofibroblast, mesenchymal stem cell, autotransplanted expandedcardiomyocyte, adipocyte, totipotent cell, pluripotent cell, blood stemcell, myoblast, adult stem cell, bone marrow cell, parenchymal cell,epithelial cell, endothelial cell, mesothelial cell, fibroblast,myofibroblast, osteoblast, chondrocyte, exogenous cell, endogenous cell,stem cell, hematopoetic stem cell, bone marrow-derived progenitor cell,progenitor cell, myocardial cell, skeletal cell, undifferentiated cell,multi-potent progenitor cell, unipotent progenitor cell, monocyte,cardiomyocyte, skeletal myoblast, macrophage, capillary endothelialcell, xenogenic cell and allogenic cell.

In some embodiments of the invention, the ECM based compositions includea protein. According to the invention, the protein can comprise, withoutlimitation, collagen, proteoglycan, glycosaminoglycan (GAG) chain,glycoprotein, cytokine and cell-surface associated protein, heparin,chondroitin sulfate B (dermatan sulfate), chondroitin sulfate A, heparansulfate, hyaluronic acid, fibronectin (Fn), tenascin, elastin,fibrillin, laminin, nidogen/entactin, fibulin I and fibulin II.

According to the invention, the pharmacological agents (or compositions)can comprise, without limitation, antiarrhythmic agents, antibiotics orantifungal agents, anti-viral agents, anti-pain agents, anesthetics,analgesics, steroidal anti-inflammatories, non-steroidalanti-inflammatories, anti-neoplastics, anti-spasmodics, modulators ofcell-extracellular matrix interactions, proteins, hormones, enzymes andenzyme inhibitors, anticoagulants and/or antithrombic agents, DNA, RNA,modified DNA and RNA, NSAIDs, inhibitors of DNA, RNA or proteinsynthesis, polypeptides, oligonucleotides, polynucleotides,nucleoproteins, compounds modulating cell migration, compoundsmodulating proliferation and growth of tissue, and vasodilating agents.

Suitable pharmacological agents and/or compositions accordingly include,without limitation, atropine, tropicamide, dexamethasone, dexamethasonephosphate, betamethasone, betamethasone phosphate, prednisolone,triamcinolone, triamcinolone acetonide, fluocinolone acetonide,anecortave acetate, budesonide, cyclosporine, FK-506, rapamycin,ruboxistaurin, midostaurin, flurbiprofen, suprofen, ketoprofen,diclofenac, ketorolac, nepafenac, lidocaine, neomycin, polymyxin b,bacitracin, gramicidin, gentamicin, oyxtetracycline, ciprofloxacin,ofloxacin, tobramycin, amikacin, vancomycin, cefazolin, ticarcillin,chloramphenicol, miconazole, itraconazole, trifluridine, vidarabine,ganciclovir, acyclovir, cidofovir, ara-amp, foscarnet, idoxuridine,adefovir dipivoxil, methotrexate, carboplatin, phenylephrine,epinephrine, dipivefrin, timolol, 6-hydroxydopamine, betaxolol,pilocarpine, carbachol, physostigmine, demecarium, dorzolamide,brinzolamide, latanoprost, sodium hyaluronate, insulin, verteporfin,pegaptanib, ranibizumab, and other antibodies, antineoplastics, AntiVGEFs, ciliary neurotrophic factor, brain-derived neurotrophic factor,bFGF, Caspase-1 inhibitors, Caspase-3 inhibitors, α-Adrenoceptorsagonists, NMDA antagonists, Glial cell line-derived neurotrophic factors(GDNF), pigment epithelium-derived factor (PEDF), and NT-3, NT-4, NGF,IGF-2.

In some embodiments of the invention, the pharmacological agentcomprises an antiarrhythmic agent selected from the group comprising,without limitation, quinidine, procainamide, disopyramide, lidocaine,phenytoin, mexiletine, flecainide, propafenone, moricizine, propranolol,esmolol, timolol, metoprolol, atenolol, amiodarone, sotalol, ibutilide,dofetilide, verapamil, diltiazem, adenosine and digoxin.

In some embodiments of the invention, the pharmacological agentcomprises an anti-inflammatory agent selected from the group comprising,without limitation, alclofenac, alclometasone dipropionate, algestoneacetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium,amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone,balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride,bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone,cliprofen, clobetasol propionate, clobetasone butyrate, clopirac,cloticasone propionate, cormethasone acetate, cortodoxone, decanoate,deflazacort, delatestryl, depo-testosterone, desonide, desoximetasone,dexamethasone dipropionate, diclofenac potassium, diclofenac sodium,diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate,diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab,enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole,fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac,flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate,flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, mesterolone,methandrostenolone, methenolone, methenolone acetate, methylprednisolonesuleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxensodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin,oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranylinehydrochloride, pentosan polysulfate sodium, phenbutazone sodiumglycerite, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate,talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam,tesimide, testosterone, testosterone blends, tetrydamine, tiopinac,tixocortol pivalate, tolmetin, tolmetin sodium, triclonide,triflumidate, zidometacin, and zomepirac sodium.

In some embodiments of the invention, the pharmacological agentcomprises a statin, i.e. a HMG-CoA reductase inhibitor. According to theinvention, suitable statins include, without limitation, atorvastatin(Lipitor®), cerivastatin, fluvastatin (Lescol®), lovastatin (Mevacor®,Altocor®, Altoprev®), mevastatin, pitavastatin (Livalo®, Pitava®),pravastatin (Pravachol®, Selektine®, Lipostat®), rosuvastatin(Crestor®), and simvastatin (Zocor®, Lipex®). Several actives comprisinga combination of a statin and another agent, such asezetimbe/simvastatin (Vytorin®), are also suitable.

As set forth in Co-pending application Ser. No. 13/782,115, Applicanthas found that the noted statins exhibit numerous beneficial propertiesthat provide several beneficial biochemical actions or activities.

According to the invention, the biologically active and pharmacologicalagents referenced above can comprise any form. In some embodiments ofthe invention, the agents, e.g. simvastatin, comprise microcapsules thatprovide delayed delivery of the agent contained therein.

Referring now to FIG. 1, there is shown a depiction of a normal humanheart 100. The heart wall 102 consists of an inner layer of simplesquamous epithelium, referred to as the endocardium. The endocardiumoverlays the myocardium (a variably thick heart muscle) and is envelopedwithin a multi-layer tissue structure referred to as the pericardium.The innermost layer of the pericardium, referred to as the visceralpericardium or epicardium, covers the myocardium. An outermost layer ofthe pericardium, referred to as the fibrous pericardium, attaches theparietal pericardium to the sternum, the great vessels and thediaphragm.

Referring now to FIG. 2, there is shown a depiction of a heart 200having an ischemic infarcted region 202, and a peri-infarcted region 204that is surrounded by healthy non-ischemic myocardium tissue 206.

As indicated above, the ischemic infarcted region 202 (or myocardialinfarction) can, and in many instances will trigger a cascading sequenceof myocellular events. In many instances, the myocellular events lead todeterioration in ventricular function and heart failure.

As discussed in detail in Co-pending application Ser. No. 13/573,569,the effects of an ischemic infarcted region can be ameliorated oreliminated by delivering an ECM based composition of the inventiondirectly to the infarcted cardiovascular tissue. In most instances, theECM based compositions will induce neovascularization, host tissueproliferation, bioremodeling, and regeneration of new cardiac tissuestructures with site-specific structural and functional properties.

According to the invention, the ECM based compositions can be deliveredto infarcted cardiovascular tissue, as well as other damaged or diseasedbiological tissue, by various conventional means. In some embodiments, amulti-needle injection system, such as disclosed in U.S. Application No.61/704,634, filed Sep. 24, 2012 and illustrated in FIGS. 3A and 3B isemployed to deliver one or more ECM based compositions to damaged ordiseased cardiovascular tissue.

Without departing from the spirit and scope of this invention, one ofordinary skill can make various changes and modifications to theinvention to adapt it to various usages and conditions. As such, thesechanges and modifications are properly, equitably, and intended to be,within the full range of equivalence of the following claims.

What is claimed is:
 1. A composition for treating a cardiovasculardisorder, comprising: an extracellular matrix (ECM) based compositioncomprising amniotic membrane and an exogenously added biologicallyactive agent, said biologically active agent comprising a growth factorselected from the group consisting of transforming growth factor-alpha(TGF-α), transforming growth factor-beta (TGF-β), fibroblast growthfactor-2 (FGF-2), basic fibroblast growth factor (bFGF) and vascularepithelial growth factor (VEGF).
 2. A composition for treating acardiovascular disorder, comprising: an extracellular matrix (ECM) basedcomposition comprising amniotic membrane and an exogenously addedbiologically active agent, said biologically active agent comprising acell selected from the group consisting of a human embryonic stem cell,fetal cardiomyocyte, myofibroblast, and mesenchymal stem cell.
 3. Acomposition for treating a cardiovascular disorder, comprising: anextracellular matrix (ECM) based composition comprising amnioticmembrane and an exogenously added biologically active agent, saidbiologically active agent comprising a cell selected from the groupconsisting of a human embryonic stem cell, fetal cardiomyocyte,myofibroblast, and mesenchymal stem cell.
 4. A composition for treatinga cardiovascular disorder, comprising: an extracellular matrix (ECM)based composition comprising amniotic membrane and a pharmacologicalagent, said pharmacological agent comprising a statin selected from thegroup consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. 5.A graft for treating a cardiovascular disorder, comprising: anextracellular matrix (ECM) based composition comprising amnioticmembrane and an exogenously added biologically active agent, saidbiologically active agent comprising a growth factor selected from thegroup consisting of transforming growth factor-alpha (TGF-α),transforming growth factor-beta (TGF-β), fibroblast growth factor-2(FGF-2), basic fibroblast growth factor (bFGF) and vascular epithelialgrowth factor (VEGF).
 6. A graft for treating a cardiovascular disorder,comprising: an extracellular matrix (ECM) based composition comprisingamniotic membrane and an exogenously added biologically active agent,said biologically active agent comprising a cell selected from the groupconsisting of a human embryonic stem cell, fetal cardiomyocyte,myofibroblast, and mesenchymal stem cell.
 7. A graft for treating acardiovascular disorder, comprising: an extracellular matrix (ECM) basedcomposition comprising amniotic membrane and an exogenously addedbiologically active agent, said biologically active agent comprising acell selected from the group consisting of a human embryonic stem cell,fetal cardiomyocyte, myofibroblast, and mesenchymal stem cell.
 8. Agraft for treating a cardiovascular disorder, comprising: anextracellular matrix (ECM) based composition comprising amnioticmembrane and a pharmacological agent, said pharmacological agentcomprising a statin selected from the group consisting of atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.
 9. A method for treating acardiovascular disorder, comprising the steps of: providing anextracellular matrix (ECM) based composition comprising amnioticmembrane and an exogenously added biologically active agent, saidbiologically active agent comprising a growth factor selected from thegroup consisting of transforming growth factor-alpha (TGF-α),transforming growth factor-beta (TGF-β), fibroblast growth factor-2(FGF-2), basic fibroblast growth factor (bFGF) and vascular epithelialgrowth factor (VEGF); and administering said ECM based composition todamaged tissue associated with said cardiovascular disorder.
 10. Amethod for treating a cardiovascular disorder, comprising the steps of:providing an extracellular matrix (ECM) based composition comprisingamniotic membrane and an exogenously added biologically active agent,said biologically active agent comprising a cell selected from the groupconsisting of a human embryonic stem cell, fetal cardiomyocyte,myofibroblast, and mesenchymal stem cell; and administering said ECMbased composition to damaged tissue associated with said cardiovasculardisorder.
 11. A method for treating a cardiovascular disorder,comprising the steps of: providing an extracellular matrix (ECM) basedcomposition comprising amniotic membrane and an exogenously addedbiologically active agent, said biologically active agent comprising acell selected from the group consisting of a human embryonic stem cell,fetal cardiomyocyte, myofibroblast, and mesenchymal stem cell; andadministering said ECM based composition to damaged tissue associatedwith said cardiovascular disorder.
 12. A method for treating acardiovascular disorder, comprising the steps of: providing anextracellular matrix (ECM) based composition comprising amnioticmembrane and a pharmacological agent, said pharmacological agentcomprising a statin selected from the group consisting of atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin; and administering said ECMbased composition to damaged tissue associated with said cardiovasculardisorder.